Purification, Gene Cloning And Expression Of A Fibrinolytic Enzyme From Douchi, A Chinese Traditional Soybean-fermented Food | Posted on:2003-10-21 | Degree:Doctor | Type:Dissertation | Country:China | Candidate:Y Peng | Full Text:PDF | GTID:1100360065460771 | Subject:Genetics | Abstract/Summary: | PDF Full Text Request | By using fibrin plate screening, fibrinolytic activity assay, SDS-PAGE analysis and thrombolytic effect test in vitro, two bacterial strains (DC-2 and DC-4) producing strongly fibrinolytic enzymes were successfully isolated from Douchi, a traditional Chinese fermented-soybean food. Strain DC-2 was identified as Bacillus subtilis and DC-4 as Bacillus amyloliquefaciens. The fibrinolytic enzyme produced by B. subtilis and B. amyloliquefaciens were named as BS-DFE and BA-DFE, respectively. B. amyloliquefaciens DC-4 was firstly found to produce a fibinolytic enzyme.The optimal fermentation conditions for producing BA-DFE were determined and the results are as follows. The compositions of fermentation medium are: 2% fibrin, 2% dextrin, 0.5% peptone, 0.15% yeast extract, 0.4% K2HPO4, 0.04% NaH2PO4, 0.3% CaCO3, pH7.0. The optimal cultivation temperature is 32?, the optimal shaking speed is 210r/min when 250mL flask contains 50mL medium, and the fermentation time is 72h. Under these conditions the fibrinolytic activity of the supernatant can reach 820 urokinase units per milliliter broth.BA-DFE was purified from the supernatant of B. amyloiquefaciens DC-4 culture broth by ammonium sulfate precipitation, ion-exchange chromatography on CM- and DEAE-Sepharose Fast Flow, hydrophobic interaction chromatography on Phenyl Sepharose 6 Fast Flow and gel filtration on Sephadex G-50. The purified enzymedisplayed thermophilic, hydrophilic and strong fibrinolytic activity. It was demonstrated that the purified BA-DFE is homogeneous by SDS-PAGE analysis and isoelectric focusing electrophoresis. The enzyme has a molecular weight of 28 kD and pI of 8.0. The optimal reaction pH value and temperature were 9.0 and 48?, respectively. BA-DFE can directly degrade the fibrin, but not activate plasminogen into plasmin and hydroly/e blood cells. It can also hydrolyze several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA. PMSF can completely inhibit its fibrinolytic activity. These results indicated that BA-DFE is a subtilisin-like serine protease, similar to nattokinase from B. natto. The first 24 amino acid residues of the N-terminal sequence of BA-DFE were AQSVPYGVSQIKAPALHSQGFTGS, which is different from those fibrinolytic enzymes, such as NK(Nattokinase), CK(a fibrinolytic enzyme from Chungkook-Jang) and KK(Katsuwokinase).Based on the high homologous N-terminal amino acid sequence between BA-DFE and Subtilisin BPN, the fragment encoding BA-DFE mature peptide was amplified from the total DNA of B. amyloliquefaciens DC-4 by PCR. The result of sequencing analysis shows that the coding region of the BA-DFE mature peptide has 825bp in length and encodes 275 amino acid residues, which has a N-terminal amino acid sequence identical to that of the purified BA-DFE determined by Edman method, indicating that the cloned fragment was indeed as expected. Sequence homologous analysis displayed that there were 80.0% and 86.5% of nucleotide and amino acid homology between BA-DFE and nattokinase gene, respectively. So these results suggested that the BA-DFE be a novel firbrinolytic enzyme.BA-DFE mature peptide-coding sequence was cloned to the restriction site Ncol and NdeI of the expression vector pET-15b, respectively, and highly expressed in E.coli BL21(DE3) by the form of non-fusion and His-BADFE fusion protein. The expressed proteins accumulated up to 40% of bacterial soluble protein after IPTG induction and formed inclusion bodies, which have no fibrinolytic activity. At present, the refolding of recombinant proteins is under investigation.Due to high homology of nucleotide between BA-DFE and subtilisin BPN, primers were designed and synthesized. The intact BA-DFE gene was amplified by PCR and cloned. The sequence analysis indicated that the BA-DFE gene has an open reading frame with 1146bp, which encodes 382 amino acid residues containing signalpeptide, pro-peptide and mature peptide. The expression plasmid called pSUGV-BADFE was constructed by inserting BA-DFE gene into E. coli-B. subtilis shuttle vector...
| Keywords/Search Tags: | Bacillus amyloliquefaciens, Bacillus subtilis, Douchi, purification, fibrinolytic enzyme, fermentation condition, gene cloning, gene expression | PDF Full Text Request | Related items |
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